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US6563583B2 - Multipass cavity for illumination and excitation of moving objects - Google Patents

Multipass cavity for illumination and excitation of moving objects Download PDF

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US6563583B2
US6563583B2 US09/976,465 US97646501A US6563583B2 US 6563583 B2 US6563583 B2 US 6563583B2 US 97646501 A US97646501 A US 97646501A US 6563583 B2 US6563583 B2 US 6563583B2
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light path
detection
illumination system
light
aperture
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US20020057432A1 (en
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William E. Ortyn
David A. Basiji
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Cytek Biosciences Inc
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Amnis LLC
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    • G02—OPTICS
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    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/144—Beam splitting or combining systems operating by reflection only using partially transparent surfaces without spectral selectivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G01N2021/052—Tubular type; cavity type; multireflective
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    • G01N2021/058—Flat flow cell

Definitions

  • This invention generally relates to illumination of moving objects or particles for purposes of analysis and detection, and more specifically, to an apparatus and method for increasing the amount of incident light upon these objects to increase scattered, fluorescent, and other signals from moving objects, such as cells, and for detecting the presence and composition of Fluorescence In-Situ Hybridization (FISH) probes within cells.
  • FISH Fluorescence In-Situ Hybridization
  • the target cells are fetal cells that cross the placental barrier into the mother's blood stream.
  • the target cells are sloughed into the blood stream from nascent cancerous tumors.
  • the target cells may be present in the blood at concentrations of one to five target cells per billion blood cells. This concentration yields only 20 to 100 cells in a typical 20 ml blood sample. In these applications, as well as others, it is imperative that the signal derived in response to the cells be as strong as possible to provide distinct features with which to discriminate the target cells from other artifacts in the sample.
  • Non-imaging cytometers take advantage of the PMT's characteristics to achieve photon-limited performance by making the illuminated area as small as possible. Decreasing the laser spot size reduces the amount of time required for an object to traverse a field of view (FOV) of the detectors. The reduced measurement time, in turn, reduces the integrated system noise, but does not reduce the signal strength of the object. The signal strength remains constant because the reduced signal integration time is balanced by the increased laser intensity in the smaller spot. For example, if the FOV in the axis parallel to flow is decreased by a factor of two, an object's exposure time will decrease by a factor of two, but the intensity at any point in that FOV will double, so the integrated photon exposure will remain constant.
  • FOV field of view
  • a common figure of merit used in flow cytometry is the coefficient of variation (CV), which equals the standard deviation of the signal over many measurements divided by the mean of the signal.
  • CV coefficient of variation
  • Photon noise increases as the mean number of photons decreases. For example, if the mean number of photons in a measurement period is four, the standard deviation will be two photons and the CV will be 50%. If the mean number of photons drops to one, the standard deviation will be one and the CV will be 100%. Therefore, to improve (i.e., decrease) the CV, the mean number of photons detected during the measurement interval must be increased.
  • One way to increase the number of photons striking the detector is to increase photon collection efficiency.
  • an alternative is to increase the number of photons emitted from the object during the measurement interval. Accordingly, it would be beneficial to provide a system in which illumination light incident on an object but not absorbed or scattered is recycled and redirected to strike the object multiple times, thereby increasing photon emission from the object.
  • a frame-based charge-coupled device (CCD) detector is used for signal detection as opposed to a PMT.
  • CCD charge-coupled device
  • the field of view along the axis of flow is approximately ten times greater than that in PMT-based flow cytometers.
  • the patent discloses a commonly used method of illumination in flow cytometry, in which the incident light is directed at the stream of particles in a direction orthogonal to the optic axis of the light collection system.
  • the method disclosed in the patent differs slightly from conventional illumination in that a highly elliptical laser spot is used, with the longer axis of the ellipse oriented along the axis of flow.
  • the entire FOV can be illuminated with laser light.
  • the intensity profile across the illuminated region has a Gaussian profile along the axis of flow. Therefore, objects at either end of the field of view will have a lower intensity of illumination light.
  • the light collection process disclosed in this patent does not continue for the duration of the full traversal of the FOV. Instead, light is collected from objects at specific regions within the FOV.
  • Object movement during the light collection process is limited to less than one pixel by use of a shutter or pulsed illumination source.
  • the illumination spot may be sized so that it substantially overfills the FOV to use an area of the Gaussian distribution near the peak where the intensity variation is minimized.
  • this approach has the undesired effect of reducing the overall intensity of illumination, or photon flux, by spreading the same amount of laser energy over a significantly larger area. The end result of reducing photon flux is a reduction in the SNR.
  • the present invention is directed to correcting beam misalignments in a multipass cavity illumination system that is adapted to increase the amount of signal emitted from an object to increase the SNR and to improve the measurement consistency of devices in which the present invention is applied.
  • a multipass cavity is formed by the placement of two mirrors on either side of a moving stream of objects.
  • a light collection system is disposed substantially orthogonal to a plane extending through the mirrors and the stream.
  • the light collection system is configured to collect light over a predefined angle and within a predefined region or field of view between the mirrors. Accordingly, the light collection system collects light that is scattered or emitted from objects as they traverse the space between the mirrors. The scattered or emitted light that is collected is directed onto a detector.
  • a light from a light source is directed through the stream of moving objects in a direction nearly orthogonal to the stream of objects but slightly inclined in the plane that extends through the mirrors and the stream. With cells and most other objects, only a small fraction of the incident light interacts with the objects via absorbance or scatter. The rest of the light passes through the stream, and is then redirected by reflection from a surface back into the stream of moving objects. The light leaves the reflecting surface at a reflected angle that is equal to an incident angle of the light. Due to the reflection angle and the distance between the stream and the first surface, the light intersects the stream on the second pass at a position that is displaced from that at which the light passed though the stream on its initial pass.
  • the light continues through the stream and is redirected by a second surface on the other side of the stream, which is substantially parallel to the first surface, back towards the stream. Again, as a result of the reflection angle and the distance between the second surface and the stream, the light passes through the stream on the third pass at a position that is displaced from that of the second pass.
  • the reflection of the light through the stream continues a plurality of times until the light has traversed a distance along the direction in which the stream is flowing that is substantially equal to the collected field of view of the light collection system. At this point, the light is no longer reflected back through the stream, but is preferably caused to exit the illumination system.
  • Beam misalignment in a multipass cavity has four degrees of freedom, position in the horizontal (X) and vertical (Y) directions and angle in the vertical and horizontal axes, termed tip and tilt respectively. Misalignments in directions along the beam axis or around the beam axis have little affect on the performance of the cavity. However, angular or positional errors lateral to the propagation direction of the beam can dramatically affect performance.
  • the present invention provides apparatus and methods to easily measure and adjust these errors in an independent fashion for each degree of freedom, in order to make beam steering corrections and maintain optical alignment.
  • the present invention of an active cavity beam detection and alignment system allows this to be accomplished in an automated, closed-loop feedback control system.
  • FIG. 1 is an isometric view of an illumination system corresponding to a first embodiment of the present invention
  • FIG. 2 (Prior Art) is an isometric view of a conventional method for illuminating objects in a flow stream;
  • FIG. 3 is an isometric view of an exemplary imaging system that implements the illumination system of FIG. 1;
  • FIG. 4 is an isometric view of an embodiment of the present invention using mirrors immersed in a fluid
  • FIG. 5 is an XY plot showing an illumination intensity profile for a conventional (Prior Art) single pass illumination scheme with a short FOV;
  • FIG. 6 is an XY plot showing an illumination intensity profile for a conventional (Prior Art) single pass illumination scheme with a tall FOV;
  • FIG. 7 is a schematic diagram illustrating a condition in which no beam overlap occurs at the center of the cavity when a smaller beam size is used in a preferred embodiment of the present invention.
  • FIG. 8 is an XY plot showing an illumination intensity profile for the embodiment of the present invention illustrated in FIG. 7, but for the case in which a narrow light beam passes through a field of view five times;
  • FIG. 9 is a schematic diagram illustrating a condition in which significant beam overlap occurs at the center of the cavity when a larger beam size is used in a preferred embodiment of the present invention.
  • FIG. 10 is an XY plot showing an illumination intensity profile for the embodiment of the present invention illustrated in FIG. 9, but for the case in which a wide light beam passes through a field of view five times;
  • FIG. 11 is a plot of the beam size in the horizontal axes for two beams with different waist sizes in a five pass embodiment of the invention, illustrating how a larger waist size can produce a smaller average beam size, thereby increasing the overall intensity of light incident on the stream;
  • FIG. 12 is an isometric view of an embodiment of the present invention that employs a retro-reflector to reverse beam traversal, increasing the number of times the beam traverses the flow stream;
  • FIGS. 13A-13F schematically illustrate an embodiment of the present invention where the beam traversal direction is reversed after a plurality of passes across the cavity by introducing an angle between the cavity mirrors;
  • FIGS. 14A-14B further illustrate schematically an embodiment of the present invention wherein the beam traversal direction is reversed after a plurality of passes across the cavity by introducing an angle between the cavity mirrors and in which the mirrors provide an optical power about an axis parallel to the flow axis for refocusing the beam in the horizontal axis with each pass through the cavity;
  • FIG. 15 is a plot of the beam size in the horizontal and vertical axes for each pass of the beam across the cavity in an embodiment employing 28 passes, as illustrated in FIGS. 14A-14B;
  • FIG. 16 schematically illustrates an embodiment of the present invention wherein the cavity mirrors have a toroidal surface profile and provide an optical power in both the horizontal and vertical axes for inducing a reversal of beam traversal direction and for refocusing the beam in both axes;
  • FIG. 17 is a plot of the beam size in the horizontal and vertical axes for each pass of the beam across the cavity in an embodiment employing 21 passes of the beam where the walls of the cavity have the toroidal surface profile illustrated in FIG. 16;
  • FIG. 18 is an isometric drawing of the beam position and angle detection system used to monitor laser alignment to the cavity in an active cavity alignment embodiment
  • FIGS. 19A-19C illustrate schematically the optical system used for active cavity alignment of the input laser beam
  • FIG. 20 is a plan view of an optical system for active cavity alignment of the input laser beam integrated with the beam detection optical system of FIG. 18;
  • FIG. 21 is a functional block diagram showing the major components of a closed-loop active cavity alignment system
  • FIG. 22 is an algorithm flow diagram illustrating the logic employed in the control system for the active cavity alignment embodiment.
  • FIG. 23 is an isometric view of an alternate embodiment of the beam position and angle detection system.
  • the present invention offers considerable advantages over the prior art for illumination of cells and other types of particles in a flow stream. These advantages arise from the recycling of laser light to increase the photon flux incident upon objects in a flow stream.
  • the present invention can also be configured to improve the uniformity of illumination, while at the same time increasing the photon flux incident upon objects, which is expected to enhance the performance of various flow cytometry applications.
  • Illumination system 10 includes a rectangular solid glass substrate 14 with reflective coatings 15 and 16 applied to two substantially parallel and flat outer surfaces 15 a and 16 a of the glass substrate.
  • a channel 20 is disposed in the rectangular solid to enable a plurality of objects 24 in a flow stream to pass through illumination system 10 between surfaces 15 a and 16 a .
  • the objects may be entrained in a sheath flow (not shown) in order to keep them centered within channel 20 .
  • a substantially cylindrical beam of light 12 such as that emitted by a laser source (not shown), is directed toward an uncoated area 13 in surface 15 a of the substrate such that a propagation axis of the beam of light (indicated by the arrow) is at a slight angle with respect to a normal to surface 15 a .
  • the beam proceeds through surface 15 a and passes through at least a portion of the plurality of objects 24 and is then reflected from reflective coating 16 back into the plurality of objects 24 .
  • the angle of propagation axis 12 a is set such that as beam of light 12 traverses the substrate, it rises a predefined amount, intersecting surface 15 a in reflective coating 15 above uncoated area 13 .
  • the beam reflects from reflective coating 15 and again passes through the plurality of objects 24 .
  • FIG. 1 As objects 24 flow along the channel, corresponding images of the objects are produced with an optical system (not shown in this Figure) having a field of view 25 .
  • an optical system (not shown in this Figure) having a field of view 25 .
  • light beam 12 continues to traverse substrate 14 such that it passes through the substrate ten times, thereby illuminating all of field of view 25 , before it is allowed to pass out of the substrate through an uncoated area 26 in surface 15 a .
  • Reflection spots 28 and dashed lines 27 illustrate the path of the light beam and indicate the points where the beam intersects and reflects from reflective coatings 15 and 16 .
  • Reflective coatings 15 and 16 form a reflection cavity 17 through which the plurality of objects 24 pass.
  • surfaces 15 and 16 could be independently mounted on their own substrates without the use of the glass substrate 14 . By reflecting the light back and forth in this manner, the total amount of light incident on objects 24 is substantially increased over that provided by conventional illumination methods.
  • FIG. 2 illustrates a common approach used in the prior art to illuminate objects in flow cytometers such as those described in U.S. Pat. No. 5,644,388.
  • an elliptical-shaped beam of light 30 is directed through the substrate 36 and passes through the plurality of objects 24 .
  • the light beam size in the flow axis is made substantially larger than that used in the present invention.
  • the intensity of light at any point in field of view 38 is substantially less than in the present invention, which reduces the amount of light scattered or otherwise emitted from the plurality of objects 24 , thereby reducing the SNR of the conventional approaches relative to the SNR of the present invention.
  • the illumination intensity varies across the field of view in accordance with a Gaussian intensity distribution of the illuminating laser light.
  • FIG. 3 shows an exemplary imaging system 40 that is substantially similar to imaging systems disclosed in copending commonly assigned U.S. Pat. No. 6,249,341, the specification and drawings of which have been specifically incorporated herein by reference.
  • the present invention is employed for illumination in imaging system 40 .
  • light 41 from an object passes through a collection lens 42 , which collects the light, producing collected light 43 .
  • the collected light is focussed substantially at infinity, i.e., the rays of collected light 43 are generally parallel and enter a prism 44 , which disperses the light, producing dispersed light 45 .
  • the dispersed light enters an imaging lens 46 , which focusses light 47 on a time-delay-integration (TDI) detector 48 .
  • TDI time-delay-integration
  • Imaging system 40 includes illumination system 10 , which was discussed above.
  • a laser light source 50 directs a beam of coherent light 51 toward a reflection cavity 52 within illumination system 10 , as shown in the Figure.
  • the illumination system may further include an aperture plate 53 , which includes an aperture 53 a having a diameter selected to reduce the size of the beam sufficiently so that the light intensity distribution across the cross section of the beam that has passed through the aperture is substantially constant.
  • the present invention may be included in other imaging systems that are described and illustrated in the above referenced copending patent application.
  • the present invention can also be configured for implementation in a stereoscopic imaging flow cytometer.
  • This configuration of the present invention is shown in FIG. 4 where a reflection cavity 59 is created by supporting two mirrors 55 and 56 on independent substrates within an immersion medium of an imaging flow cytometer.
  • the ends of two capillary tubes 64 a and 64 b are brought within close proximity to each other.
  • a stream of objects 63 is hydrodynamically focused with capillary tube 64 a and caused to flow through a gap 64 c between the tubes and into capillary tube 64 b.
  • Two water immersion objectives 61 and 66 are mounted on a frame (not shown) and are employed to image the gap between the capillary tubes onto two pixilated detectors 62 and 67 .
  • Mirrors 55 and 56 which are supported within the immersion cavity on a frame 60 , create reflection cavity 59 around the stream of objects 63 .
  • Light from an illumination source (not shown) is directed along a path 65 under mirror 55 , through stream of objects 63 , and onto mirror 56 .
  • the light is redirected back through stream of objects 63 , and caused to again traverse the stream of objects, generally in the manner described above, in regard to FIG. 1 .
  • the first prior art system to be discussed is incorporated in a widely-available, non-imaging commercial flow cytometer system.
  • This system employs a 15 mW continuous wave laser that produces an elliptical beam spot 70 microns wide by 20 microns tall, a 6 m/s sample flow rate, and a PMT detector (not shown).
  • An intensity profile along a flow path of the illumination system is illustrated in FIG. 5 .
  • the profile has a peak intensity 107 that is approximately 0.68 photons/microsecond through the area defined by the absorbance cross section of a fluorescein molecule.
  • the intensity varies over the field of view of the collection system in accordance with a Gaussian distribution function, 1/e 2x , wherein “x” is a ratio of the distance along the traversal path to the radius of the beam.
  • x is a ratio of the distance along the traversal path to the radius of the beam.
  • the boundaries of a Gaussian beam are defined at a 1/e 2 point 108 , which is the position at which the intensity falls to approximately 13% of the peak intensity.
  • each fluorescein molecule emits an average of 1.29 photons as it traverses the illuminated region.
  • the emission of photons is quantized (no fractional photons are emitted) and that some molecules emit no photons, while others emit one or more photons when traversing the illuminated region.
  • the resulting average number of emissions per molecule over all molecules is a fractional number.
  • the second prior art example is the same as the first except that the dimension of the illuminating beam is 500 microns in the axis parallel to the direction of object flow.
  • FIG. 6 illustrates an intensity profile for the enlarged illumination area produced by this second prior art system.
  • a peak intensity 109 for this profile is approximately 0.027 photons/microsecond, which is 25 times lower than in the first example shown in FIG. 5 .
  • the average emission per fluorescein molecule remains 1.29 photons due to the increased illumination time allowed by the taller beam.
  • there is no difference in the average emission per fluorescein dye molecule in the two prior art systems there is no change in instrument performance, despite the 25-fold change in beam height. Changes in the beam height along the axis of the flow stream do not change the number of fluorescent photons emitted by the sample as it flows through the illuminated region, because the increased illumination time is offset by a corresponding reduced photon flux per unit area.
  • FIG. 7 illustrates an embodiment of the present invention wherein the beam height is 100 microns in the axis parallel to flow, and the beam is reflected across the illuminated region five times.
  • the beam incident angle is inclined relative to the reflecting surfaces so that there is no overlap of the beam in the center of the cavity.
  • the resulting total illuminated height is therefore 500 microns, like that of the second prior art example discussed just above.
  • the beam width is increased from the 70 micron dimension in the prior art, to 90 microns in order to reduce beam divergence.
  • the average number of photons emitted per dye molecule is increased to 4.78 photons, more than a factor of three greater than is obtained using conventional illumination in the prior art.
  • the increase in emitted photons is a result of two factors: (1) high illumination flux due to compact beam dimensions; and (2) an extended illumination height (and correspondingly longer illumination time), due to the multiple offset passes of the laser beam through the illumination region.
  • FIG. 8 The intensity profile along the stream axis, which provides the increased illumination flux of the above embodiment, is illustrated in FIG. 8 . From FIG. 8, it is apparent that a five-pass embodiment produces a peak intensity 138 of more than 0.10 photons/microsecond through the area defined by the absorbance cross section of the fluorescein molecule, which is four times greater than that shown in FIG. 6 for the prior art illumination configuration with the same illumination height.
  • the increase in intensity of the exciting beam and the increase in the number of passes in which the exciting beam encounters a molecule in the present invention produce more fluorescence from each molecule of the dye.
  • the present invention enables control of the illumination intensity profile in the cavity at the intersection of the stream and each of the plurality of beam passes.
  • an advantageous illumination profile may be achieved.
  • FIG. 9 shows a configuration similar to that of FIG. 7, except that the beam height is increased to produce a 50% overlap between beam segments in adjacent passes.
  • FIG. 10 shows the resulting intensity profile, which is of much higher uniformity than is produced under no-overlap conditions.
  • the extent of beam overlap from pass to pass can be controlled by modifying the distance between the cavity's reflective surfaces and by changing the incident angle of the beam. Increasing the distance between the reflective surfaces and/or the incident angle allows the beam to propagate farther along the vertical axis between passes across the center of the cavity.
  • beam overlap can occur as a result of a divergence of the beam as it traverses the cavity. Divergence due to diffraction causes the cross-sectional area of the beam to increase as the beam traverses the cavity. As the traversal distance increases, a concomitant increase in cross-sectional beam area, or beam spread occurs. This increase in beam spread decreases the intensity, or photon flux at any given portion in the cross section of the beam, which in turn, reduces the probability of fluorescence excitation of probe molecules. Therefore, the beam spread must be kept within acceptable limits.
  • the beam waist i.e., the point of the smallest cross-sectional area of the beam
  • the beam cross-sectional size increases in either direction away from the waist at a rate that is inversely proportional to the size of the waist. This phenomenon is illustrated in FIG. 11, which shows the spread of two beams over five passes across the center of a 5 mm wide cavity, one beam having a 50 micron waist (line 141 with triangles at data points) and the other an 80 micron waist (line 143 with squares at data points).
  • the waist size may be chosen appropriately to maximize intensity based on the number of cavity traversals and the acceptable beam size at points away from the waist, or in regard to the average beam size within the cavity.
  • the beam waist may be disposed appropriately within or outside the cavity to achieve a desired effect with the present invention.
  • various parameters can be adjusted to increase the number of cavity traversals the beam makes while maintaining a beam size that is appropriate to increase fluorescence.
  • the cavity may be made narrower to decrease the path length that the beam must travel as it traverses the cavity. In this manner, the number of passes in the cavity can be increased while still maintaining a small cross-sectional beam size and thereby maintaining relatively high beam intensity.
  • FIG. 12 shows an embodiment of illumination system 10 in accord with the present invention in which a retro-reflector 139 is included to reflect the beam back into the cavity after it has exited the top of the cavity.
  • the embodiment shown in this Figure is substantially identical to the first preferred embodiment shown in FIG. 1 .
  • retro-reflector 139 reflects the beam back along the path it followed before exiting the cavity, so that the beam reversing its previous path through the substrate.
  • This embodiment effectively doubles the number of beam passes through the cavity achieved by the embodiment in FIG. 1.
  • a 20-pass retro-reflected embodiment will provide nearly a 15-fold increase in the average photon exposure of an object over a conventional single-pass illumination.
  • FIGS. 13A-13F illustrate another embodiment of the present invention wherein the beam traverses the cavity and reverses direction, but unlike the embodiment of FIG. 12, it does so without the use of a retro-reflector.
  • an angle is introduced between two surfaces 15 a′ and 16 a′, which comprise the walls of the cavity.
  • the angle between the reflecting surfaces is exaggerated and shown as equal to ten degrees.
  • the introduction of the angle between the two surfaces causes a gradual reduction in the incident angle of the beam relative to the surfaces, as the beam repeatedly traverses the cavity. Eventually, the incident angle becomes 90 degrees, or reverses sign, and the beam is reflected back upon itself (or down the walls of the cavity) and re-traverses the cavity in the opposite direction.
  • the cross-sectional beam size converges to a minimum at the waist position then diverges.
  • the intensity of the beam at any point is inversely proportional to the square of the beam diameter.
  • the embodiment of the present invention shown in FIGS. 14A and 14B incorporates optical power in the reflecting surfaces of the cavity.
  • Each wall of the cavity is a cylindrical mirror 151 and 153 with curvature in the horizontal plane selected to focus the light beam that is reflected therefrom within the cavity.
  • each wall's radius of curvature, R is the flow stream, so with each reflection of the light beam, the diverging beam is refocused by the mirrors on the objects within the flow stream.
  • a small beam diameter is maintained in the vicinity of the flow stream, and the beam spread in the axis perpendicular to flow is minimized so that more light is focused on the objects in the stream than could otherwise be obtained.
  • the embodiment shown in FIGS. 14A and 14B also incorporates the method of beam reversal illustrated in FIGS. 13A-F.
  • the size of a 488 nm laser beam waist in the vertical and horizontal axes for this embodiment is plotted in FIG. 15 .
  • the beam size in the axis perpendicular to flow is maintained at 40 microns in the vicinity of the flow stream.
  • the beam diameter alternately converges on the flow stream and then diverges toward the reflecting surface where, upon reflection, the beam re-converges near the flow stream.
  • the cylindrical surface contains no optical power in the axis of flow. Therefore, the beam diameter upon the first intersection with the stream is 199 microns. The beam continues to converge up to the 14 th pass where the beam waist, or minimum beam diameter, of 91 microns is reached.
  • a flow cytometer employing this embodiment with a 28-pass cavity, produces an average photon emission per dye molecule of 44.32 photons. This result represents a 35-fold increase in signal strength compared to the conventional method of illumination, where only a 1.29 photon per molecule average emission is achieved.
  • FIG. 16 illustrates an alternative embodiment of the present invention where the reflection cavity surfaces 161 and 163 are toroids with a radius of 50 mm about an axis perpendicular to the page and a radius of approximately 1 mm about the axis along the flow stream.
  • the centers of curvature for the 50 mm surfaces are separated by approximately 98 mm, so that the vertex of each mirror is separated by 2 mm and centered on the flow stream axis.
  • the illumination beam enters the reflective cavity perpendicular to the flow stream axis at a point approximately 1 mm below the axis defined by a line running between the centers of curvature for the two 50 mm surfaces.
  • the beam waist is located within the reflective cavity and the beam makes a first flow stream intersection.
  • the beam traverses the cavity and is reflected upward at an angle of approximately 2.3 degrees from horizontal, causing the beam to re-cross the cavity and strike the other wall of the cavity.
  • the beam reflects from this cavity wall at an angle of about 4.4 degrees with respect to horizontal and continues to re-cross the cavity and strike the opposite surface in this manner such that the reflected angle with the horizontal increases upon each reflection of the beam by one of the surfaces.
  • the beam traverses the cavity and crosses the axis defined by a line 165 running between the centers of curvature of the two 50 mm radii surfaces 161 and 163 . At this point, the normals to these surfaces point downward. Therefore, the reflected angle of the beam with respect to the horizontal decreases.
  • the reflection angle of the beam with respect to the horizontal is approximately 8.1 degrees.
  • the reflection angle is reduced to approximately 7.4 degrees.
  • the beam makes an angle of approximately zero degrees to the horizontal, and after striking the other wall, the propagation direction of the beam with respect to the flow axis is reversed. The beam then propagates down the cavity, reflecting from the surfaces and eventually exits the cavity at its point of entry after making twenty two passes through the flow stream.
  • FIG. 17 illustrates the beam waist size during the propagation of a 488 nm laser beam through the embodiment of the invention illustrated in FIG. 16, where the reflecting surfaces have optical power in both axes.
  • the beam intersects the flow stream on the first pass with a 50 micron waist in each axis. After the beam passes through the stream it begins to diverge and strikes the far wall of the cavity. Upon reflection, the beam re-converges in the vertical plane such that the waist is approximately 50 microns when it crosses the flow stream. As described in the previous embodiment the beam always re-converges at the flow stream with a waist size of 50 microns in the vertical plane after striking the cavity wall. However, in the axis parallel to flow the beam continues to diverge after reflecting off the cavity wall.
  • the optical power in that axis is insufficient to cause the beam to re-converge. Therefore, when the beam intersects the flow axis on the second pass, it is approximately 55 microns in the axis parallel to flow.
  • the optical power in the axis parallel to flow reduces the divergence from what it would be if the surface contained no optical power in the that axis, but the divergence continues to increase as the beam enters the far field propagation regime.
  • the beam waist is approximately 176 microns. From this point on the beam begins to converge back toward a 50 micron waist, but exits the cavity before reaching a dimension of 50 microns in the axis parallel to flow.
  • the present invention can be equipped with an active beam detection and alignment system, to maintain optical alignment of the input laser beam to the multipass cavity.
  • Beam misalignment in a multipass cavity has four degrees of freedom, position in the horizontal (X) and vertical (Y) directions and angle in the vertical and horizontal axes, termed tip and tilt respectively. That definition of tip and tilt should be applied to the following disclosure and the claims that follow.
  • FIG. 18 illustrates a beam detection system 212 that can be used to quantify both the positional and angular error of a beam of light exiting the cavity of a flow cell. Further, the error for all four degrees of freedom can be quantified independently.
  • the embodiment shown in FIG. 18 depicts the arrangement for a beam of light leaving the cavity through a cavity exit aperture 170 . However the same principles can be applied for a portion of the beam of light split off from the main beam before entering the cavity, as is shown in FIG. 20 .
  • the beam of light After the cavity through exit aperture 170 , it passes through a lens element 172 , which is followed by an amplitude beam splitter 174 , which splits the beam into two optical paths, according to the beam splitter coating transmission to reflection ratio T/R.
  • T/R ratio is 50/50 to provide approximately 50% of the incident light into each optical path.
  • the reflected beam is directed onto a position sensitive detector 176 , such as a quadrant detector or quad cell.
  • the transmitted beam passes through lens element 178 , and is then directed onto a second quad cell 180 .
  • Lens element 172 is positioned such that its front focal plane is located at the cavity exit aperture 170
  • the first quad cell (Angle Quad Cell), position sensitive detector 176 is placed behind a rear focal plane of lens 172 .
  • position sensitive detector 176 is shifted off of an optical axis 190 by beam splitter 174 .
  • a beam 182 exiting cavity exit aperture 170 with an angular error 184 will be imaged by lens 172 laterally displaced from the center of position sensitive detector 176 .
  • exit aperture 170 is at the front focal plane of lens 172 , all beams of the same angle will be focussed at the same location on position sensitive detector 176 , independent of their position in cavity exit aperture 170 .
  • the Be angular error changes horizontally and vertically, the focused beam position on the quad cell will change in both the X and Y direction.
  • the Angle Quad Cell determines the tip and tilt error in the beam.
  • Lens element 178 is positioned to receive the light transmitted through beam splitter 174 .
  • the Position Quad Cell (second quad cell 180 ) is located at the rear focal plane of lens 178 .
  • an image of cavity exit aperture 170 is formed on the Position Quad Cell (second quad cell 180 ) with a lateral magnification determined by the ratio of focal lengths of lens element 178 to lens element 172 .
  • the magnification of the system can be made greater than 1 to increase the sensitivity of position error detection at the Position Quad Cell (second quad cell 180 ). Therefore, the Position Quad Cell can be used to measure the positional error of the beam at cavity exit aperture 170 .
  • positional error includes both the lateral error as described above, as well as any error along the Y axis of cavity exit aperture 170 (an up or down deviation from the center axis of cavity exit aperture 170 ).
  • positional error encompasses non-angular errors, including errors about both the X axis of cavity exit aperture 170 (a lateral deviation from the center axis of cavity exit aperture 170 ) and the Y axis of cavity exit aperture 170 (an up or down deviation from the center axis of cavity exit aperture 170 ).
  • FIG. 18 illustrates a magnification of unity for exit aperture 170 imaged onto second quad cell 180 (the Position Quad Cell). In practice the magnification will be greater than unity. In one embodiment a magnification of 5 ⁇ is employed by using focal lengths of 50 mm and 250 mm for lenses 172 and 178 , respectively.
  • Active cavity beam detection system 212 of FIG. 18 allows for the measurement of beam misalignment to the multipass cavity in an independent fashion for each degree of freedom X, Y, tip and tilt. To facilitate active alignment of an input laser beam to the multipass cavity it is desirable to have independent control over these parameters in the input optical system.
  • FIGS. 19A, 19 B, and 19 C each show plan views of an optical system 210 that provides for independent control of the beam position and angle at the input aperture of the multipass cavity.
  • system 210 is shown in an aligned condition.
  • the collimated (e.g., light rays substantially focussed to infinity) input laser beam 192 is redirected by a first reflective element 194 , placed at the front focal plane of a lens 196 , and is focused at the rear focal plane of a lens 196 , where a second reflective element 198 is disposed.
  • Reflective element 198 is also located at the front focal plane of a lens 200 .
  • multipass cavity aperture 202 After redirection by reflective element 198 , the laser beam is recollimated by lens 200 , before entering a multipass cavity aperture 202 , which is located at the rear focal plane of lens 200 .
  • multipass cavity aperture 202 is effectively collimated by lens 200 at reflective element 198 .
  • Multipass cavity aperture 202 is also imaged by lens 196 onto reflective element 194 .
  • first reflecting element 194 also referred to as the Angle Adjustment Mirror, is adjusted in angle to produce a positional change of the beam at second reflecting element 198 .
  • This positional change of the beam at the front focal plane of lens 200 results in an angular change of an input beam 204 at cavity input aperture 202 .
  • the Angle Adjustment Mirror (first reflecting element 194 ), is shown in the aligned position with the laser beam centered on second reflective element 198 , which is also referred to as the Position Adjusting Mirror.
  • the Position Adjustment Mirror (second reflective element 198 ), is adjusted in angle, and because it is at the front focal plane of lens 200 , it produces only a positional change in beam 206 at cavity input aperture 202 . Therefore, independent control of all four degrees of freedom X, Y, tip and tilt are provided by this optical system.
  • FIG. 20 is a plan view of an integrated beam detection and alignment system in which active cavity beam detection system 212 of FIG. 18 has been added to the beam alignment optical system 210 of FIGS. 19A-C, by employing a beam splitter.
  • beam splitter 208 is disposed in the optical path before the entrance to the multipass cavity, and samples the laser light as it exits the cavity.
  • the beam splitter ratio T/R is chosen so as to maximize the transmission of the input laser beam, while providing sufficient reflected light to ensure adequate signal to noise for the beam detection system.
  • T/R is chosen so as to maximize the transmission of the input laser beam, while providing sufficient reflected light to ensure adequate signal to noise for the beam detection system.
  • FIG. 21 is a functional block diagram showing the major components of a closed-loop active cavity alignment system.
  • light from beam alignment optical system 210 is sampled and imaged onto the paired quad cell detectors of active cavity beam detection system 212 .
  • the Angle Quad Cell measures the angle of the beam as it exits multipass cavity aperture 202 .
  • the Position Quad Cell (second quad cell 180 ) measures the position of the beam as it exits multipass cavity aperture 202 .
  • a Beam Alignment Processor 214 analyzes the output from Beam Detection System 212 , and measures the error in the beam position or angle as it leaves multipass cavity aperture 202 .
  • a System Controller 216 retrieves the measurements from Beam Alignment Processor 214 and corrects angular and positional errors in laser input beam 192 by employing Motorized Mirror Mounts 220 and a Motor Driver 218 to effect the required adjustments to beam alignment optical system 210 .
  • Motorized mirror mounts incorporate motorized actuators that adjust the mirror angle in two axes, tip and tilt.
  • processor 214 and controller 216 can be either a computing device (such as a personal computer) or a logical circuit (such as an application specific integrated circuit (ASIC)).
  • ASIC application specific integrated circuit
  • FIG. 22 illustrates a flow chart showing the sequence of logical steps in a method for actively controlling beam alignment optical system 210 , to ensure the precise alignment of the beam to multipass cavity aperture 202 .
  • Beam Alignment Controller 214 reads the output of Angle Quad Cell (position sensitive detector 176 ) in a block 222 , and converts that value into a measurement of the angular error of beam, relative to the optical axis of the multipass cavity aperture 202 , in a block 224 .
  • Angle Quad Cell position sensitive detector 176
  • Beam Alignment Controller 214 calculates a corrected position for the Angle Adjustment Mirror (first reflecting element 194 ) that eliminates the error in the angle of the beam as it leaves the cavity in a block 228 .
  • System Controller 216 queries the measurements and moves the Angle Adjustment Mirror (first reflecting element 194 ) to the new position to eliminate the detected error, in a block 230 .
  • Beam Alignment Processor 214 reads the output of Position Quad Cell (position sensitive detector 176 ) in a block 232 converts the value into a measurement of the positional error of beam relative to the optical axis of the cavity, in a block 234 .
  • decision block 226 if it is determined that the angular error does not exceed a predefined limit, then the logic moves from block 226 directly to block 232 , and the logic reads the output of Position Quad Cell as described above.
  • the positional error is compared to a predetermined threshold. If the error is greater than the predetermined threshold, Beam Alignment Processor 214 calculates a corrected position for Position Adjustment Mirror (second reflective element 198 ) that eliminates the error in the position of the beam as it leaves the cavity, in a block 238 . Once the corrected position is determined, System Controller 216 moves the Position Adjustment Mirror (second reflective element 198 ) to the new position to eliminate the detected error, in a block 240 . The method is then complete. If in decision block 236 it is determined that the error determined in block 234 does not exceed the predetermined threshold, the method is similarly completed.
  • FIG. 23 An alternate embodiment of the beam detection system is illustrated in FIG. 23 .
  • two elongate mirrors 242 and 244 are used to reflect the beam back and forth to increase the path length of the beam, before it strikes the Angle Quad Cell (position sensitive detector 176 ).
  • the Angle Quad Cell position sensitive detector 176
  • This embodiment does not decouple the angular and positional errors on each quad cell.
  • imaging lens 178 is used to image cavity exit aperture 170 at the Position Quad Cell (second quad cell 180 ) as previously described, then the Position Quad Cell (second quad cell 180 ) measures the position error directly, and this can be subtracted from the Angle Quad Cell measurement to determine the angular error.
  • beam alignment optical system 210 and all other optical systems described herein, it will be understood that the lenses and other optical elements illustrated are shown only in a relatively simple form.
  • focusing lenses 196 and 200 are illustrated as compound lenses with two elements, such as cemented achromatic doublets, lens elements of different designs, either simpler or more complex, could be used in constructing the imaging system to provide the desired optical performance, as will be understood by those of ordinary skill in the art.
  • Lens elements may be conventional ground and polished or molded glass or plastic lenses with spherical or aspheric surfaces, reflective optics with power or diffractive optical elements.
  • Compound optical systems may include both transmissive and reflective optics of any of the above type.
  • beam splitter optical components such as beam splitter 208 may be of the plate type as illustrated, with the beam splitter optical coating deposited on one surface; or the cube type, with the coating surface bonded between two prism elements, or the pellicle type.
  • Reflective elements 194 and 198 may be plane mirrors as shown with metallic, dielectric, or hybrid (metallic and dielectric) type optical coatings deposited on the mirror surface.
  • Prism elements may also be used for reflecting the beam, employing total internal reflection from uncoated surfaces, or with reflective optical coatings of the metallic, dielectric, or hybrid type deposited on the reflective face of the prism.
  • CMOS complementary metal-oxide semiconductors

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